Processes and systems for metabolite production using hydrogen rich c1-containing substrates

ABSTRACT

The invention is directed to a process for producing one or more fermentation product in a multi-stage process including an inoculation reactor and at least one bioreactor. The inoculation reactor is fed a C1-containing gaseous substrate containing a reduced amount of hydrogen. The hydrogen is reduced to increase the proportion of CO in the C1-containing gaseous substrate being provided to the inoculation reactor. The inoculation reactor ferments the CO-rich C1-containing gaseous substrate and produces an inoculum, which is fed to at least one bioreactor. The bioreactor receives the C1-containing gaseous substrate, which may or may not contain reduced amounts of hydrogen, to produce one or more fermentation product. By providing a CO-rich C1-containing gaseous substrate to the inoculation reactor, both the inoculation reactor and the subsequent bioreactor(s), are able to have increased stability and product selectivity.

CROSS-REFERENCE TO A RELATED APPLICATION

The application is a divisional of U.S. application Ser. No. 16/123,464filed on Sep. 6, 2018, which claims the benefit of U.S. ProvisionalApplication No. 62/556,099 filed Sep. 8, 2017, both of which areincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a process for producing one or morefermentation product through a multi-stage gas fermentation processincluding an inoculation reactor and at least one bioreactor. Inparticular, the invention relates to a process whereby a CO-richC1-containing gaseous substrate is fed to the inoculation reactor toproduce an inoculum.

BACKGROUND OF THE INVENTION

Carbon dioxide (CO₂) accounts for about 76% of global greenhouse gasemissions from human activities, with methane (16%), nitrous oxide (6%),and fluorinated gases (2%) accounting for the balance (the United StatesEnvironmental Protection Agency). Reduction of greenhouse gas emissions,particularly CO₂, is critical to halting the progression of globalwarming and the accompanying shifts in climate and weather.

It has long been recognized that catalytic processes, such as theFischer-Tropsch process, may be used to convert gases containing carbondioxide (CO₂), carbon monoxide (CO), and/or hydrogen (H₂), such asindustrial waste gas or syngas, into a variety of fuels and chemicals.Recently, however, gas fermentation has emerged as an alternativeplatform for the biological fixation of such gases. In particular,C1-fixing microorganisms have been demonstrated to convert gasescontaining CO₂, CO, and/or H₂ into products such as ethanol and2,3-butanediol.

Such gasses may be derived, for example, from industrial processes,including gas from carbohydrate fermentation, gas from cement making,pulp and paper making, steel making, oil refining and associatedprocesses, petrochemical production, coke production, anaerobic oraerobic digestion, synthesis gas (derived from sources including but notlimited to biomass, liquid waste streams, solid waste streams, municipalstreams, fossil resources including natural gas, coal and oil), naturalgas extraction, oil extraction, metallurgical processes, for productionand/or refinement of aluminium, copper, and/or ferroalloys, geologicalreservoirs, and catalytic processes (derived from steam sourcesincluding but not limited to steam methane reforming, steam naphthareforming, petroleum coke gasification, catalyst regeneration—fluidcatalyst cracking, catalyst regeneration-naphtha reforming, and drymethane reforming).

With particular industrial processes, the composition of the gas may notbe ideal for fermentation. When the composition of the gas is not ideal,cell growth, product selectivity, and stability may be less thanoptimal.

Accordingly, there remains a need for an invention which optimizes thecomposition of gas from industrial processes to promote cell growth,product selectivity, and stability in a downstream fermentation process.

BRIEF SUMMARY OF THE INVENTION

The invention provides a process for producing one or more fermentationproduct, wherein a CO-rich C1-containing gaseous substrate is passed toan inoculation reactor comprising a liquid nutrient medium containing aculture of one or more C1-fixing microorganism where the CO-richC1-containing gaseous substrate is fermented to produce an inoculum, atleast a portion of the inoculum is passed to a bioreactor system, thebioreactor system defining at least one bioreactor containing a cultureof one or more C1-fixing microorganism in a liquid nutrient medium, anH₂ rich C1-containing gaseous substrate is passed to the bioreactorsystem where the H₂ rich C1-containing gaseous substrate is fermented toproduce at least one fermentation product.

In particular embodiments, the CO-rich C1-containing gaseous substratebeing passed to the inoculation reactor comprises H₂ at an H₂:CO molarratio of less than 1:1.

In certain instances, the CO-rich C1-containing gaseous substrate beingpassed to the inoculation reactor comprises H₂ at an H₂:CO molar ratioof less than 0.5:1.

Preferably, the CO rich C1-containing gaseous substrate being passed tothe inoculation reactor comprises H₂ at a H₂:CO molar ratio between0.02:1 to 1:1. In certain embodiments, the H₂:CO molar ratio is between0.05:1 to 1:1, or 0.15:1 to 1:1, or 0.25:1 to 1:1, or 0.35:1 to 1:1, or0.45:1 to 1:1, or 0.55:1 to 1:1, or 0.65:1 to 1:1, or 0.75:1 to 1:1, or0.85:1 to 1:1, or 0.95:1 to 1:1. 0012 In particular embodiments, the H₂rich C1-containing gaseous substrate being passed to the bioreactorsystem comprises H₂ at an H₂:CO molar ratio of at least 1.1:1.

Preferably, the H₂ rich C1-containing gaseous substrate being passed tothe bioreactor system comprises H₂ at an H₂:CO molar ratio between 1.1:1to 6:1. In certain embodiments, the H₂:CO molar ratio is between 1.5:1to 6:1, or 2:1 to 6:1, or 2.5:1 to 6:1, or 3:1 to 6:1, or 3.5:1 to 6:1,or 4:1 to 6:1, or 4.5:1 to 6:1, or 5:1 to 6:1.

In at least one embodiment, the C1-fixing microorganism in the either,or both, the inoculation reactor or the bioreactor system is acarboxydotrophic bacterium.

In embodiments where the C1-fixing microorganism is carboxydotrophic,the carboxydotrophic bacterium may be selected from the group consistingof Moorella, Clostridium, Ruminococcus, Acetobacterium, Eubacterium,Butyribacterium, Oxobacter, Methanosarcina, and Desulfotomaculum.

Preferably, the carboxydotrophic bacterium is Clostridiumautoethanogenum.

In at least one embodiment, the bioreactor system comprises one or moreprimary bioreactors linked to one or more secondary bioreactors.

Preferably, the process provides for the passing of at least a portionof a C1-containing gaseous substrate to an inoculation reactor and atleast a portion of the C1-containing gaseous substrate to a bioreactor,where the C1-containing gaseous substrate in the inoculation reactor isfermented to produce an inoculum, where at least a portion of theinoculum is passed to at least one bioreactor where the C1-containinggaseous substrate in the bioreactor is fermented to produce at least onefermentation product, and wherein the C1-containing gaseous substratebeing passed to the inoculation reactor is subjected to at least one H₂removal process prior to being passed to the inoculation reactor.

In particular embodiments, the C1-containing gaseous substrate beingpassed to the inoculation reactor comprises H₂ at an H₂:CO molar ratioof less than 1:1.

In certain instances, the C1-containing gaseous substrate being passedto the inoculation reactor comprises H₂ at an H₂:CO molar ratio of lessthan 0.8:1.

In certain instances, the C1-containing gaseous substrate being passedto the inoculation reactor comprises H₂ at an H₂:CO molar ratio of lessthan 0.5:1.

Preferably, the C1-containing gaseous substrate being passed to theinoculation reactor comprises H₂ at a H₂:CO molar ratio between 0.02:1to 1:1. In certain embodiments, the H₂:CO molar ratio is between 0.05:1to 1:1, or 0.15:1 to 1:1, or 0.25:1 to 1:1, or 0.35:1 to 1:1, or 0.45:1to 1:1, or 0.55:1 to 1:1, or 0.65:1 to 1:1, or 0.75:1 to 1:1, or 0.85:1to 1:1, or 0.95:1 to 1:1.

In certain embodiments, the H₂ removal process comprises at least onepressure swing adsorption process.

In certain embodiments, the H₂ removal process comprises at least onemembrane separation module.

Preferably, at least a portion of the C1-containing gaseous substrate isderived from an industrial source.

In certain instances, at least a portion of the C1-containing gaseoussubstrate may be derived from at least one industrial source selectedfrom the group consisting of carbohydrate fermentation, gasfermentation, cement making, pulp and paper making, steel making, oilrefining and associated processes, petrochemical production, cokeproduction, anaerobic or aerobic digestion, synthesis gas, natural gasextraction, oil extraction, metallurgical processes, for productionand/or refinement of aluminium, copper, and/or ferroalloys, geologicalreservoirs, and catalytic processes.

Preferably, the process produces at least one fermentation productselected from the group consisting of: ethanol, acetate, butanol,butyrate, 2,3-butanediol, 1,3-butanediol, lactate, butene, butadiene,methyl ethyl ketone, ethylene, acetone, isopropanol, lipids,3-hydroxypropionate, isoprene, fatty acids, 2-butanol, 1,2-propanediol,1-propanol, monoethylene glycol, isobutene, and C6-C14 alcohols.

In at least one embodiment, the one or more fermentation product isfurther converted to at least one component of diesel fuel, jet fuel,gasoline, propylene, nylon 6-6, rubber, and/or resins.

In particular embodiments, at least one fermentation product ismicrobial biomass. In certain instances, this microbial biomass may befurther processed to produce at least one component of animal feed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram depicting the integration of ahydrogen removal process, an inoculation reactor, and a bioreactorsystem.

FIG. 2 is a schematic flow diagram depicting the integration of ahydrogen removal process, an inoculation reactor, and a bioreactorsystem, where the hydrogen removal process is upstream of both theinoculation reactor and the bioreactor system, in accordance with oneaspect of the invention.

FIG. 3 is a schematic flow diagram further depicting two water-gas-shiftprocesses and a pressure swing adsorption process upstream of thebioreactor system, where one water-gas-shift process is bypassed, inaccordance with one aspect of the invention.

FIG. 4 is a schematic flow diagram further depicting two water-gas-shiftprocesses and a pressure swing adsorption process upstream of thebioreactor system, in accordance with one aspect of the invention.

FIG. 5 is a schematic flow diagram further depicting additional hydrogenremoval processes upstream of the inoculation reactor, in accordancewith one aspect of the invention.

FIGS. 6a and 6b are graphs showing metabolite production and gas uptakein a first bioreactor according to Example 1.

FIGS. 7a and 7b are graphs showing metabolite production and gas uptakein a second bioreactor according to Example 1.

FIGS. 8a and 8b are graphs showing metabolite production and gas uptakein a first bioreactor according to Example 2.

FIGS. 9a and 9b are graphs showing metabolite production and gas uptakein a second bioreactor according to Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified that by optimizing the composition of agas stream being fed to the inoculation reactor, cell growth, productselectivity, and stability are optimized in both the inoculation reactorand subsequent bioreactor system. In particular, the inventors havefound optimal cell growth, product selectivity, and stability when thegas stream being fed to the inoculation reactor comprises a reducedamount of hydrogen.

Definitions

Unless otherwise defined, the following terms as used throughout thisspecification are defined as follows:

“C1” refers to a one-carbon molecule, for example, CO, CO₂, CH₄, orCH₃OH. “C1-oxygenate” refers to a one-carbon molecule that alsocomprises at least one oxygen atom, for example, CO, CO₂, or CH₃OH.“C1-carbon source” refers a one carbon-molecule that serves as a partialor sole carbon source for the microorganism of the invention. Forexample, a C1-carbon source may comprise one or more of CO, CO₂, CH₄,CH₃OH, or CH₂O₂. Preferably, the C1-carbon source comprises one or bothof CO or CO₂. A “C1-fixing microorganism” is a microorganism that hasthe ability to produce one or more products from a C1-carbon source.Typically, the microorganism of the invention is a C1-fixingmicroorganism.

“C1-containing gaseous substrates” include any gas leaving theindustrial process comprising C1. In various instances, theC1-containing gaseous substrate comprises CO, H₂, CO₂, or combinationsthereof. The gaseous substrate will typically contain a significantproportion of CO, preferably at least about 5% to about 100% CO byvolume. The gaseous substrate may contain a significant proportion ofhydrogen. For example, in particular embodiments, the substrate maycomprise an approximately 2:1, or 1:1, or 1:2 ratio of H₂:CO. In oneembodiment, the substrate comprises about 30% or less H₂ by volume, 20%or less H₂ by volume, about 15% or less H₂ by volume or about 10% orless H₂ by volume. The substrate may also contain some CO₂ for example,such as about 1% to about 80% CO₂ by volume, or 1% to about 30% CO₂ byvolume. In one embodiment, the substrate comprises less than or equal toabout 20% CO₂ by volume. In particular embodiments, the substratecomprises less than or equal to about 15% CO₂ by volume, less than orequal to about 10% CO₂ by volume, less than or equal to about 5% CO₂ byvolume or substantially no CO₂. Additionally, the C1-containing gaseoussubstrate may contain one or more of oxygen (O₂), nitrogen (N₂), and/ormethane (CH₄).

Although the substrate is typically gaseous, the substrate may also beprovided in alternative forms. For example, the substrate may bedissolved in a liquid saturated with a CO-containing gas using amicrobubble dispersion generator. By way of further example, thesubstrate may be adsorbed onto a solid support.

The term “co-substrate” refers to a substance that, while notnecessarily being the primary energy and material source for productsynthesis, can be utilized for product synthesis when added to anothersubstrate, such as the primary substrate.

The substrate and/or C1-carbon source may be a waste gas obtained as aby-product of an industrial process or from some other source, such asfrom automobile exhaust fumes or biomass gasification. In certainembodiments, the industrial process is selected from the groupconsisting gas emissions from carbohydrate fermentation, gasfermentation, gas emissions from cement making, pulp and paper making,steel making, oil refining and associated processes, petrochemicalproduction, coke production, anaerobic or aerobic digestion, synthesisgas (derived from sources including but not limited to biomass, liquidwaste streams, solid waste streams, municipal streams, fossil resourcesincluding natural gas, coal and oil), natural gas extraction, oilextraction, metallurgical processes, for production and/or refinement ofaluminium, copper, and/or ferroalloys, geological reservoirs, andcatalytic processes (derived from the steam sources including but notlimited to steam methane reforming, steam naphtha reforming, petroleumcoke gasification, catalyst regeneration—fluid catalyst cracking,catalyst regeneration-naphtha reforming, and dry methane reforming). Inthese embodiments, the substrate and/or C1-carbon source may be capturedfrom the industrial process before it is emitted into the atmosphere,using any convenient method.

“Gas stream” refers to any stream of substrate which is capable of beingpassed, for example, from one module to another, from one module to abioreactor, from one module to an inoculation reactor, from one processto another process, and/or from one module to a carbon capture means.

The term “carbon capture” as used herein refers to the sequestration ofcarbon compounds including CO₂ and/or CO from a stream comprising CO₂and/or CO and either:

converting the CO₂ and/or CO into products; or

converting the CO₂ and/or CO into substances suitable for long-termstorage; or

trapping the CO₂ and/or CO in substances suitable for long-term storage;

or a combination of these processes.

“Reactants” as used herein refer to a substance that takes part in andundergoes change during a chemical reaction. In particular embodiments,the reactants include, but are not limited to, CO and/or H₂.

“Hydrogen removal process” and the like includes technologies that arecapable of removing and/or separating hydrogen from the C1-containinggaseous substrate. In particular embodiments, a pressure swingadsorption process and/or a membrane separation process are used as thehydrogen removal process.

The term “bioreactor” “bioreactor system” and the like includes afermentation device consisting of one or more vessels and/or towers orpiping arrangements, which includes the Continuous Stirred Tank Reactor(CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR),Bubble Column, Gas Lift Fermenter, Static Mixer, a circulated loopreactor, a membrane reactor, such as a Hollow Fibre Membrane Bioreactor(HFM BR) or other vessel or other device suitable for gas-liquidcontact. The bioreactor is preferably adapted to receive a gaseoussubstrate comprising CO or CO₂ or H₂ or mixtures thereof. The bioreactormay comprise multiple reactors (stages), either in parallel or inseries. Preferably, the bioreactor is configured to receive an inoculumfrom an invocation reactor. Preferably, the bioreactor is configured asa production reactor, where most of the fermentation products areproduced.

The term “inoculation reactor”, “inoculator”, “seed reactor” and thelike includes a fermentation device for establishing and promoting cellgrowth. The inoculation reactor is preferably adapted to receive agaseous substrate comprising CO or CO₂ or H₂ or mixtures thereof.Preferably, the inoculation reactor is a reactor where cell growth isfirst initiated. In various embodiments, the inoculation reactor iswhere previously growth cells are revived. In the various embodiments,the inoculator initiates cell growth of one or more microorganism toproduce an inoculum, which may then be transferred to the bioreactorsystem where each bioreactor is configured to promote the production ofone or more fermentation product. In certain instances, the inoculatorhas a reduced volume when compared to the subsequent one or morebioreactor.

“Nutrient media” or “Nutrient medium” is used to describe bacterialgrowth media. Generally, this term refers to a media containingnutrients and other components appropriate for the growth of a microbialculture. The term “nutrient” includes any substance that may be utilizedin a metabolic pathway of a microorganism. Exemplary nutrients includepotassium, B vitamins, trace metals, and amino acids.

The term “fermentation broth” or “broth” is intended to encompass themixture of components including nutrient media and a culture or one ormore microorganisms. It should be noted that the term microorganism andthe term bacteria are used interchangeably throughout the document.

The term “inoculum” is intended to encompass the fermentation brothinitially grown in the inoculation reactor which is then passed to theone or more subsequent bioreactors to seed the one or more subsequentbioreactor. Preferably, the inoculum is utilized by the one or morebioreactors to produce one or more fermentation product.

The term “desired composition” is used to refer to the desired level andtypes of components in a substance, such as, for example, of a gasstream. More particularly, a gas is considered to have a “desiredcomposition” if it contains a particular component (e.g. CO, H₂, and/orCO₂) and/or contains a particular component at a particular proportionand/or does not contain a particular component (e.g. a constituentharmful to the microorganisms) and/or does not contain a particularcomponent at a particular proportion. More than one component may beconsidered when determining whether a gas stream has the desiredcomposition. In one or more embodiment, the “desired composition” of theC1-containing gaseous substrate is defined in terms of an H₂:CO molarratio. In various embodiments, the desired composition of theC1-containing gaseous substrate being passed to the inoculation reactordiffers from the desired composition of the C1-containing gaseoussubstrate being passed to the bioreactor system.

The terms “increasing the efficiency”, “increased efficiency” and thelike, when used in relation to a fermentation process, include, but arenot limited to, increasing one or more of the rate of growth ofmicroorganisms catalysing the fermentation, the growth and/or productproduction rate at elevated product concentrations, the volume ofdesired product produced per volume of substrate consumed, the rate ofproduction or level of production of the desired product, and therelative proportion of the desired product produced compared with otherby-products of the fermentation.

Unless the context requires otherwise, the phrases “fermenting”,“fermentation process” or “fermentation reaction” and the like, as usedherein, are intended to encompass both the growth phase and productbiosynthesis phase of the gaseous substrate.

A “microorganism” is a microscopic organism, especially a bacterium,archea, virus, or fungus. The microorganism of the invention istypically a bacterium. As used herein, recitation of “microorganism”should be taken to encompass “bacterium.”

A “parental microorganism” is a microorganism used to generate amicroorganism of the invention. The parental microorganism may be anaturally-occurring microorganism (e.g., a wild-type microorganism) or amicroorganism that has been previously modified (e.g., a mutant orrecombinant microorganism). The microorganism of the invention may bemodified to express or overexpress one or more enzymes that were notexpressed or overexpressed in the parental microorganism. Similarly, themicroorganism of the invention may be modified to contain one or moregenes that were not contained by the parental microorganism. Themicroorganism of the invention may also be modified to not express or toexpress lower amounts of one or more enzymes that were expressed in theparental microorganism. In one embodiment, the parental microorganism isClostridium autoethanogenum, Clostridium ljungdahlii, or Clostridiumragsdalei. In a preferred embodiment, the parental microorganism isClostridium autoethanogenum LZ1561, which was deposited on Jun. 7, 2010,with Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)located at Inhoffenstraß 7B, D-38124 Braunschweig, Germany on Jun. 7,2010, under the terms of the Budapest Treaty and accorded accessionnumber DSM23693. This strain is described in International PatentApplication No. PCT/NZ2011/000144, which published as WO 2012/015317.

The term “derived from” indicates that a nucleic acid, protein, ormicroorganism is modified or adapted from a different (e.g., a parentalor wild-type) nucleic acid, protein, or microorganism, so as to producea new nucleic acid, protein, or microorganism. Such modifications oradaptations typically include insertion, deletion, mutation, orsubstitution of nucleic acids or genes. Generally, the microorganism ofthe invention is derived from a parental microorganism. In oneembodiment, the microorganism of the invention is derived fromClostridium autoethanogenum, Clostridium ljungdahlii, or Clostridiumragsdalei. In a preferred embodiment, the microorganism of the inventionis derived from Clostridium autoethanogenum LZ1561, which is depositedunder DSMZ accession number DSM23693.

“Wood-Ljungdahl” refers to the Wood-Ljungdahl pathway of carbon fixationas described, i.e., by Ragsdale, Biochim Biophys Acta, 1784: 1873-1898,2008. “Wood-Ljungdahl microorganisms” refers, predictably, tomicroorganisms containing the Wood-Ljungdahl pathway. Generally, themicroorganism of the invention contains a native Wood-Ljungdahl pathway.Herein, a Wood-Ljungdahl pathway may be a native, unmodifiedWood-Ljungdahl pathway or it may be a Wood-Ljungdahl pathway with somedegree of genetic modification (e.g., overexpression, heterologousexpression, knockout, etc.) so long as it still functions to convert CO,CO₂, and/or H₂ to acetyl-CoA.

An “anaerobe” is a microorganism that does not require oxygen forgrowth. An anaerobe may react negatively or even die if oxygen ispresent above a certain threshold. However, some anaerobes are capableof tolerating low levels of oxygen (e.g., 0.000001-5 vol. % oxygen).Typically, the microorganism of the invention is an anaerobe.

“Acetogens” are obligately anaerobic bacteria that use theWood-Ljungdahl pathway as their main mechanism for energy conservationand for the synthesis of acetyl-CoA and acetyl-CoA-derived products,such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008).In particular, acetogens use the Wood-Ljungdahl pathway as a (1)mechanism for the reductive synthesis of acetyl-CoA from CO₂, (2)terminal electron-accepting, energy conserving process, (3) mechanismfor the fixation (assimilation) of CO₂ in the synthesis of cell carbon(Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3^(rd) edition, p.354, New York, N.Y., 2006). All naturally occurring acetogens areC1-fixing, anaerobic, autotrophic, and non-methanotrophic. Typically,the microorganism of the invention is an acetogen.

An “ethanologen” is a microorganism that produces or is capable ofproducing ethanol. Typically, the microorganism of the invention is anethanologen.

An “autotroph” is a microorganism capable of growing in the absence oforganic carbon. Instead, autotrophs use inorganic carbon sources, suchas CO and/or CO₂. Typically, the microorganism of the invention is anautotroph.

A “carboxydotroph” is a microorganism capable of utilizing CO as a solesource of carbon and energy. Typically, the microorganism of theinvention is a carboxydotroph.

The microorganism of the invention may be cultured with the gas streamto produce one or more products. For instance, the microorganism of theinvention may produce or may be engineered to produce ethanol (WO2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO2012/053905), butyrate (WO 2008/115080), 2,3-butanediol (WO 2009/151342and WO 2016/094334), lactate (WO 2011/112103), butene (WO 2012/024522),butadiene (WO 2012/024522), methyl ethyl ketone (2-butanone) (WO2012/024522 and WO 2013/185123), ethylene (WO 2012/026833), acetone (WO2012/115527), isopropanol (WO 2012/115527), lipids (WO 2013/036147),3-hydroxypropionate (3-HP) (WO 2013/180581), terpenes, includingisoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO2013/185123), 1,2-propanediol (WO 2014/036152), 1-propanol (WO2014/0369152), chorismate-derived products (WO 2016/191625),3-hydroxybutyrate (WO 2017/066498), and 1,3-butanediol (WO2017/0066498). In addition to one or more target products, themicroorganism of the invention may also produce ethanol, acetate, and/or2,3-butanediol. In certain embodiments, microbial biomass itself may beconsidered a product. These products may be further converted to produceat least one component of diesel, jet fuel, and/or gasoline.Additionally, the microbial biomass may be further processed to producea single cell protein (SCP).

A “single cell protein” (SCP) refers to a microbial biomass that may beused in protein-rich human and/or animal feeds, often replacingconventional sources of protein supplementation such as soymeal orfishmeal. To produce a single cell protein or other product, the processmay comprise additional separation, processing, or treatments steps. Forexample, the method may comprise sterilizing the microbial biomass,centrifuging the microbial biomass, and/or drying the microbial biomass.In certain embodiments, the microbial biomass is dried using spraydrying or paddle drying. The method may also comprise reducing thenucleic acid content of the microbial biomass using any method known inthe art, since intake of a diet high in nucleic acid content may resultin the accumulation of nucleic acid degradation products and/orgastrointestinal distress. The single cell protein may be suitable forfeeding to animals, such as livestock or pets. In particular, the animalfeed may be suitable for feeding to one or more beef cattle, dairycattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo/bison,llamas, alpacas, reindeer, camels, bantengs, gayals, yaks, chickens,turkeys, ducks, geese, quail, guinea fowl, squabs/pigeons, fish, shrimp,crustaceans, cats, dogs, and rodents. The composition of the animal feedmay be tailored to the nutritional requirements of different animals.Furthermore, the process may comprise blending or combining themicrobial biomass with one or more excipients.

An “excipient” may refer to any substance that may be added to themicrobial biomass to enhance or alter the form, properties, ornutritional content of the animal feed. For example, the excipient maycomprise one or more of a carbohydrate, fiber, fat, protein, vitamin,mineral, water, flavor, sweetener, antioxidant, enzyme, preservative,probiotic, or antibiotic. In some embodiments, the excipient may be hay,straw, silage, grains, oils or fats, or other plant material. Theexcipient may be any feed ingredient identified in Chiba, Section 18:Diet Formulation and Common Feed Ingredients, Animal Nutrition Handbook,3^(rd) revision, pages 575-633, 2014.

A “native product” is a product produced by a genetically unmodifiedmicroorganism. For example, ethanol, acetate, and 2,3-butanediol arenative products of Clostridium autoethanogenum, Clostridium ljungdahlii,and Clostridium ragsdalei. A “non-native product” is a product that isproduced by a genetically modified microorganism but is not produced bya genetically unmodified microorganism from which the geneticallymodified microorganism is derived.

“Selectivity” refers to the ratio of the production of a target productto the production of all fermentation products produced by amicroorganism. The microorganism of the invention may be engineered toproduce products at a certain selectivity or at a minimum selectivity.In one embodiment, a target product account for at least about 5 wt. %,10 wt. %, 15 wt. %, 20 wt. %, 30 wt. %, 50 wt. %, 75 wt. %, or 90 wt. %of all fermentation products produced by the microorganism of theinvention. In one embodiment, the target product accounts for at least10 wt. % of all fermentation products produced by the microorganism ofthe invention, such that the microorganism of the invention has aselectivity for the target product of at least 10 wt. %. In anotherembodiment, the target product accounts for at least 30 wt. % of allfermentation products produced by the microorganism of the invention,such that the microorganism of the invention has a selectivity for thetarget product of at least 30 wt. %. In one embodiment, the targetproduct accounts for at least 90 wt. % of all fermentation productsproduced by the microorganisms, such that the microorganism of theinvention has a selectivity for the target product of at least 90 wt. %.

Target products may be separated or purified from a fermentation brothusing any method or combination of methods known in the art, including,for example, fractional distillation, evaporation, pervaporation, gasstripping, phase separation, and extractive fermentation, including, forexample, liquid-liquid extraction. In certain embodiments, targetproducts are recovered from the fermentation broth by continuouslyremoving a portion of the broth from the bioreactor, separatingmicrobial cells from the broth (conveniently by filtration), andrecovering one or more target products from the broth. Alcohols and/oracetone may be recovered, for example, by distillation. Acids may berecovered, for example, by adsorption on activated charcoal. Separatedmicrobial cells are preferably returned to the bioreactor. The cell-freepermeate remaining after target products have been removed is alsopreferably returned to the bioreactor. Additional nutrients (such as Bvitamins) may be added to the cell-free permeate to replenish the mediumbefore it is returned to the bioreactor.

The culture/fermentation should desirably be carried out underappropriate conditions for production of the target product. Typically,the culture/fermentation is performed under anaerobic conditions.Reaction conditions to consider include pressure (or partial pressure),temperature, gas flow rate, liquid flow rate, media pH, media redoxpotential, agitation rate (if using a continuous stirred tank reactor),inoculum level, maximum gas substrate concentrations to ensure that gasin the liquid phase does not become limiting, and maximum productconcentrations to avoid product inhibition. In particular, the rate ofintroduction of the substrate may be controlled to ensure that theconcentration of gas in the liquid phase does not become limiting, sinceproducts may be consumed by the culture under gas-limited conditions.

Operating a bioreactor at elevated pressures allows for an increasedrate of gas mass transfer from the gas phase to the liquid phase.Accordingly, it is generally preferable to perform theculture/fermentation at pressures higher than atmospheric pressure.Also, since a given gas conversion rate is, in part, a function of thesubstrate retention time and retention time dictates the required volumeof a bioreactor, the use of pressurized systems can greatly reduce thevolume of the bioreactor required and, consequently, the capital cost ofthe culture/fermentation equipment. This, in turn, means that theretention time, defined as the liquid volume in the bioreactor dividedby the input gas flow rate, can be reduced when bioreactors aremaintained at elevated pressure rather than atmospheric pressure. Theoptimum reaction conditions will depend partly on the particularmicroorganism used. However, in general, it is preferable to operate thefermentation at a pressure higher than atmospheric pressure. Also, sincea given gas conversion rate is in part a function of substrate retentiontime and achieving a desired retention time, in turn, dictates therequired volume of a bioreactor, the use of pressurized systems cangreatly reduce the volume of the bioreactor required, and consequentlythe capital cost of the fermentation equipment.

DESCRIPTION

Controlling the composition of the C1-containing gaseous substrate beingfed to an inoculator and/or a bioreactor has been found particularlyuseful for promoting cell growth, product selectivity, and stabilityboth in the inoculation reactor and subsequent bioreactors. Preferably,the C1-containing substrate is composition controlled before being fedto an inoculation reactor, to produce an inoculum for feeding one ormore downstream reactors. Preferably, the inoculation reactor comprisesa culture of one or more C1-fixing microorganism in a liquid nutrientmedium and is capable of receiving the composition controlledC1-containing gaseous substrate to produce an inoculum throughfermentation.

The inventors have found that when a C1-containing gaseous substratethat is rich in hydrogen is used for fermentation, the fermentationprocess often lacks long-term product selectivity and stability.Surprisingly, the inventors have found that when operating afermentation process under hydrogen-rich conditions, providing analternate carbon monoxide (CO) rich C1-containing stream to theinoculation reactor, results in not only an increase in biomass growthand biomass growth rate, but also results in increased selectivity toethanol and improved stability in the downstream bioreactors.

This invention has particular applicability to fermentation processesutilizing industrial gas streams comprising H₂ at a H₂:CO molar ratio ofat least 3:1, however it is considered that the invention is alsobeneficial to industrial streams comprising lower H₂ compositions suchas gas streams having H₂:CO molar ratios of 2:1 or 1.5:1, or 1.1:1. Inone embodiment, the invention provides a process for producing one ormore fermentation product, the process comprising: (a) passing at leasta portion of a C1-containing gaseous substrate to an inoculation reactorand at least a portion of the C1-containing gaseous substrate to abioreactor; (b) fermenting the C1-containing gaseous substrate in theinoculation reactor to produce an inoculum; (c) passing at least aportion of the inoculum to at least one bioreactor; and (d) fermentingthe C1-containing gaseous substrate in the bioreactor to produce atleast one fermentation product; wherein the C1-containing gaseoussubstrate being passed to the inoculation reactor is subjected to atleast one H₂ removal process prior to being passed to the inoculationreactor.

In particular embodiments, the bioreactor comprises one or more primaryreactors linked to one or more secondary reactors. In certainembodiments, the primary reactor(s) operates at conditions to promotebiomass production, and the secondary reactor(s) operates at conditionsto promote metabolite production. In various embodiments, the H₂ richC1-containing gaseous substrate provided to the primary and secondaryreactors is from the same industrial source and has substantially thesame composition.

In one embodiment, the CO-rich C1-containing gaseous substrate, and theH₂ rich C1-containing gaseous substrate are derived from the sameindustrial source. In various embodiments, at least a portion of an H₂rich C1-containing gaseous substrate is passed to a hydrogen removalprocess, prior to being provided to the inoculation reactor, thehydrogen removal process being configured to separate at least a portionof hydrogen from the H₂ rich C1-containing gaseous substrate to producethe CO-rich C1-containing gaseous substrate. In particular embodiments,the treatment zone comprises an H₂ membrane separation module and/or apressure swing adsorption (PSA) process. Preferably, the hydrogenremoval process comprises a membrane separation module.

In one or more embodiment, the H₂ rich C1-containing gaseous substrateis derived from an industrial process.

In alternative embodiments, the CO-rich C1-containing gaseous substratecomprises a bottled CO gas stream. In one embodiment, the bottled CO gasis blended with one or more gaseous components such as nitrogen and/orcarbon dioxide. In further embodiments, the CO-rich C1-containinggaseous substrate is a CO-rich gaseous stream derived from a differentsource than the H₂ rich C1-containing gaseous substrate. In oneembodiment, the CO-rich C1-containing gaseous substrate is derived froma CO₂ electrolysis process.

Hydrogen Separation

The volume of gas necessary may, in some instances, make using bottledgas prohibitive due to cost. Therefore, it is preferred that the H₂ richC1-containing gaseous substrate is treated to remove at least a portionof hydrogen from the substrate and produce a CO-rich C1-containinggaseous substrate. Suitable methods for treating an H₂ richC1-containing gaseous substrate may include but are not limited to,membrane separation technologies, and pressure swing adsorptiontechnologies.

Membrane separation modules provide a low cost, relatively simple way toremove at least a portion of hydrogen from a gaseous substrate. Forexample, a reformer syngas with the composition of 72 vol. % H₂, 14 vol.% CO, 7 vol. % CO₂ and 7 vol. % CH₄ at 25 bara pressure passing througha demonstrative membrane separation module results in a high-pressureCO-rich stream and a low-pressure H₂ rich stream. The high-pressureCO-rich stream remains at 25 bara and contains 50 vol. % CO, 16 vol. %H₂, 25 vol. % CH₄, and 9 vol. % CO₂. The low-pressure H₂ rich stream isreduced to 1 bara and contains 92 vol. % H₂, 6 vol. % CO₂, and 1 vol. %each of CO and CH₄. The high-pressure CO-rich stream can be provided tothe inoculator as a CO-rich C1-containing gaseous substrate. Thehigh-pressure CO-rich stream provides the added benefit of not requiringfurther compression, thus avoiding the capital cost associated with anaddition compressor unit for the inoculation reactor.

Pressure swing adsorption process technologies are a more complicatedyet effective way to remove at least a portion of hydrogen from agaseous substrate. When utilizing a pressure swing adsorption process,the resulting CO-rich stream is low pressure. While the use of apressure swing adsorption process is feasible, the CO-rich C1-containinggaseous substrate may need to be compressed prior to being provided tothe inoculation reactor or any bioreactor, thereby increasing thecapital cost associated with the inoculation reactor. This may be atleast partially offset, however, when considering the fact that thehydrogen stream produced by the pressure swing adsorption process is athigh pressure and can be sold as a product.

CO₂ Electrolysis

An alternative method for providing a CO-rich C1-containing gaseoussubstrate is through use of CO₂ electrolysis. CO₂ electrolysis processesconvert a CO₂ feedstock to CO and O₂. The use of a CO₂ electrolysisprocess to provide a CO-rich stream for the inoculator may be ofinterest at industrial sites comprising a CO₂ rich stream in addition toan O₂ rich stream. Additionally, it is further considered that the tailgas from the inoculation reactor and/or the bioreactor system, beingrich in CO₂ can be used as a feedstock for the CO₂ electrolysis units.

FIG. 1 shows a schematic flow diagram of one embodiment of theinvention. A portion of a C1-containing gaseous substrate is passed viapiping means 110 to an inoculation reactor 130 where the C1-containingsubstrate is fermented to produce an inoculum. At least a portion of theinoculum is passed via piping means 131 to the bioreactor system 140,150where a portion of the C1-containing gaseous substrate is also passedvia piping means 110 to be fermented to produce at least one product141, 151. The C1-containing gaseous substrate being passed to theinoculation reactor 130 is subjected to at least one hydrogen removalprocess 120 before being sent to the inoculation reactor 130. Thehydrogen removal process 120 receives the C1-containing gaseoussubstrate via piping means 110 and removes at least a portion of thehydrogen 121 from the C1-containing gaseous substrate to produce aCO-rich C1-containing gaseous substrate, which is fed to the inoculationreactor 130 via piping means 122.

Preferably, the C1-containing gaseous substrate being passed to theinoculation reactor 130 comprises H₂ at an H₂:CO molar ratio of lessthan 1:1. In certain embodiments the C1-containing gaseous substratebeing passed to the inoculation reactor 130 comprises H₂ at an H₂:COmolar ratio of less than 0.8:1. Preferably, the C1-containing gaseoussubstrate being passed to the inoculation reactor 130 comprises H₂ at anH₂:CO molar ratio between 0.02:1 to 1:1. In various instances, thehydrogen removal process 120 removes at least a portion of hydrogenthrough use of at least one membrane separation module. In variousinstances, the hydrogen removal process 120 removes at least a portionof hydrogen through use of at least one pressure swing adsorptionprocess. In various embodiments, the hydrogen removal process 120removes at least a portion of hydrogen through use of both a membraneseparation module and a pressure swing adsorption process.

In certain instances, the C1-containing gaseous substrate being fed tothe inoculation reactor 130 and the bioreactor system 140, 150 isderived at least in part from an industrial source. Preferably, theindustrial source is selected from the group consisting of carbohydratefermentation, gas fermentation, cement making, pulp and paper making,steel making, oil refining and associated processes, petrochemicalproduction, coke production, anaerobic or aerobic digestion, synthesisgas, natural gas extraction, oil extraction, metallurgical processes,for production and/or refinement of aluminium, copper, and/orferroalloys, geological reservoirs, and catalytic processes.

Preferably, the fermentation product 141, 151 produced by the bioreactorsystem 140, 150 is selected from the group consisting of: ethanol,acetate, butanol, butyrate, 2,3-butanediol, 1,3-butanediol, lactate,butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol,lipids, 3-hydroxypropionate, isoprene, fatty acids, 2-butanol,1,2-propanediol, 1-propanol, monoethylene glycol, isobutene, and C6-C14alcohols. In various instances, at least a portion of the product 141,151 is further converted to at least one component of diesel fuel, jetfuel, gasoline, propylene, nylon 6-6, rubber, and/or resins. In variousinstances, at least one fermentation product 141, 151 is microbialbiomass. This microbial biomass may, in some instances, be furtherprocessed to produce at least one component of animal feed.

In various embodiments, the fermentation broth from one bioreactor 140may be passed to another bioreactor 150 within the bioreactor system140,150 via piping means 142.

FIG. 2 shows a schematic flow diagram of one embodiment of theinvention. A portion of a C1-containing gaseous substrate is passed viapiping means 210 to an inoculation reactor 230 where the C1-containingsubstrate is fermented to produce an inoculum. At least a portion of theinoculum is passed via piping means 231 to the bioreactor system 240,250where a portion of the C1-containing gaseous substrate is also passedvia piping means 210 to be fermented to produce at least one product241, 251. The C1-containing gaseous substrate being passed to theinoculation reactor 230 and the bioreactor system 140,150 is subjectedto at least one hydrogen removal process 220 before being sent to theinoculation reactor 230. The hydrogen removal process 220 receives theC1-containing gaseous substrate via piping means 210 and removes atleast a portion of the hydrogen 221 from the C1-containing gaseoussubstrate to produce a CO-rich C1-containing gaseous substrate, which isfed to the inoculation reactor 230 via piping means 222 and to thebioreactor system, 140,150 via piping means 223.

Preferably, the C1-containing gaseous substrate being passed to theinoculation reactor 230 and the bioreactor system 240,250 comprises H₂at an H₂:CO molar ratio of less than 1:1. In certain embodiments theC1-containing gaseous substrate being passed to the inoculation reactor230 and the bioreactor system 240,250 comprises H₂ at an H₂:CO molarratio of less than 0.8:1. Preferably, the C1-containing gaseoussubstrate being passed to the inoculation reactor 230 and the bioreactorsystem 240,250 comprises H₂ at an H₂:CO molar ratio between 0.02:1 to1:1. In various instances, the hydrogen removal process 220 removes atleast a portion of hydrogen through use of at least one membraneseparation module. In various instances, the hydrogen removal process220 removes at least a portion of hydrogen through use of at least onepressure swing adsorption process. In various embodiments, the hydrogenremoval process 220 removes at least a portion of hydrogen through useof both a membrane separation module and a pressure swing adsorptionprocess.

In certain instances, the C1-containing gaseous substrate being fed tothe inoculation reactor 230 and the bioreactor system 240, 250 isderived at least in part from an industrial source. Preferably, theindustrial source is selected from the group consisting of carbohydratefermentation, gas fermentation, cement making, pulp and paper making,steel making, oil refining and associated processes, petrochemicalproduction, coke production, anaerobic or aerobic digestion, synthesisgas, natural gas extraction, oil extraction, metallurgical processes,for production and/or refinement of aluminium, copper, and/orferroalloys, geological reservoirs, and catalytic processes.

Preferably, the fermentation product 241, 251 produced by the bioreactorsystem 240, 250 is selected from the group consisting of: ethanol,acetate, butanol, butyrate, 2,3-butanediol, 1,3-butanediol, lactate,butene, butadiene, methyl ethyl ketone, ethylene, acetone, isopropanol,lipids, 3-hydroxypropionate, isoprene, fatty acids, 2-butanol,1,2-propanediol, 1-propanol, monoethylene glycol, isobutene, and C6-C14alcohols. In various instances, at least a portion of the product 241,251 is further converted to at least one component of diesel fuel, jetfuel, gasoline, propylene, nylon 6-6, rubber, and/or resins. In variousinstances, at least one fermentation product 241, 251 is microbialbiomass. This microbial biomass may, in some instances, be furtherprocessed to produce at least one component of animal feed.

In various embodiments, the fermentation broth from one bioreactor 240may be passed to another bioreactor 250 within the bioreactor system240,250 via piping means 242.

FIGS. 3, 4 and 5 depict various embodiments of the invention, using ahydrogen production process of a refining operation as the industrialsource of the H₂ rich C1-containing gaseous substrate. A typicalhydrogen production process, as depicted in FIG. 3, FIG. 4 and FIG. 5,contains the following stages: (i) a reforming process wherein a CH₄containing feedstock is converted to a syngas stream comprising CO andH₂; (ii) at least one water gas shift step, wherein a portion of the COis reacted with water to produce H₂ and CO₂; and (iii) a pressure swingadsorption (PSA) module adapted to recover hydrogen from the gas stream.

FIG. 3 shows one embodiment of the invention utilizing an H₂ richC1-containing gaseous substrate from a reforming process 310. At least aportion of the H₂ rich C1-containing gaseous substrate is flowed to amembrane separation module 350 via piping means 312. The membraneseparation module 350 separates the C1-containing gaseous substrate intoa high-pressure CO-rich stream, and a low pressure H₂ rich stream. Atleast a portion of the low-pressure CO-rich stream is passed to aninoculation reactor 370 via piping means 352. At least a portion of thelow-pressure H₂ rich stream is passed to a pressure swing adsorptionprocess 360 via piping means 351. In at least one embodiment, thegaseous substrate is passed to a compressor prior to being passed to thepressure swing adsorption process 360. In one embodiment, the CO-richstream comprises at least 40% CO, or at least 50% CO, or at least 60%CO. In one embodiment the pressure of the CO-rich C1-containing streamis at least 15 bar, or at least 20 bar, or at least 25 bar.

In various embodiments, the process may include multiple water gas shiftprocesses 320, 330 and/or multiple hydrogen removal processes 350, 340,360. As shown in FIG. 3, the C1-containing gaseous substrate may firstbe passed from a reforming process 310 to a water gas shift process 320via piping means 311 to convert at least a portion of the CH₄ to asyngas stream comprising CO and H₂. This gas stream may optionallybypass one or more further water gas shift process 330 via piping means321 and be fed to the one or more hydrogen removal process 340 toseparate at least a portion of the hydrogen 341 from the gas stream.This stream may then be passed to one or more further hydrogen removalprocess 360 via piping means 342. The stream from the one or morefurther hydrogen removal process 360 may be sent to the bioreactor 380via piping means 361 for fermentation. At least a portion of thesubstrate not sent to the bioreactor may optionally be sent to thereforming process 310 via piping means 362. In various instances, thebioreactor 380 receives the gaseous substrate and produces one or morefermentation product 381. Optionally, the tail gas from both theinoculation reactor 370 and the bioreactor 380 can be passed back to thereforming process 310 via separate piping means 372, 382 and/or ablended stream 378.

In the various embodiments, the inoculation reactor 370 and thebioreactor 380 are configured in a step-wise manner, whereby theinoculation reactor 370 ferments a CO-rich C1-containing gaseoussubstrate to produce an inoculum, which is then fed to the bioreactor380 via piping means 371. By utilizing this inoculum in the bioreactor380, product selectivity and stability of the fermentation process isimproved.

In another embodiment, as shown in FIG. 4, an H₂ rich C1-containingstream from a reforming process 410 is flowed to pressure swingadsorption process 450 via piping means 412 provided upstream of theinoculation reactor 470. The pressure swing adsorption process 450separates the C1-containing stream into a high-pressure H₂ rich streamand a low-pressure CO-rich stream. The low-pressure CO-rich stream maybe passed to a compressor prior to being passed to the inoculationreactor 470 via piping means 452. In one embodiment, the CO-rich streambeing passed to the inoculation reactor 470 comprises at least 30% CO orat least 40% CO, or at least 50% CO, or at least 60% CO. The separatedhydrogen may be passed from the pressure swing adsorption process 450 toanother pressure swing adsorption process 460 via piping means 451. Invarious embodiments, the process may include multiple water gas shiftprocesses 420, 430 and/or multiple hydrogen removal processes 450, 440,460.

As shown in FIG. 4, the C1-containing gaseous substrate may first bepassed from a reforming process 410 to a water gas shift process 420 viapiping means 411 to convert at least a portion of the CH₄ to a syngasstream comprising CO and H₂. This gas stream may then be passed to oneor more further water gas shift process 430 via piping means 421 and befed to the one or more hydrogen removal process 440 via piping means 431to separate at least a portion of the hydrogen 441 from the gas stream.This stream may then be passed to one or more further hydrogen removalprocess 460 via piping means 442. The stream from the one or morefurther hydrogen removal process 460 may be sent to the bioreactor 480via piping means 461 for fermentation. At least a portion of thesubstrate not sent to the bioreactor may optionally be sent to thereforming process 410 via piping means 462. In various instances, thebioreactor 480 receives the gaseous substrate and produces one or morefermentation product 481. Optionally, the tail gas from both theinoculation reactor 470 and the bioreactor 480 can be passed back to thereforming process 410 via separate piping means 472, 482 and/or ablended stream 478.

In the various embodiments, the inoculation reactor 470 and thebioreactor 480 are configured in a step-wise manner, whereby theinoculation reactor 470 ferments a CO-rich C1-containing gaseoussubstrate to produce an inoculum, which is then fed to the bioreactor480 via piping means 471. By utilizing this inoculum in the bioreactor480, product selectivity and stability of the fermentation process isimproved.

In another embodiment, as shown in FIG. 5, the C1-containing stream fromthe reforming process 510 may be sent to multiple hydrogen removalprocesses 540, 550, 560, 590 before being sent to either the inoculationreactor 570 and/or the bioreactor 580. In various instances, theC1-containing stream may be sent to a compressor before and/or between ahydrogen removal process. By sending the C1-containing stream tomultiple hydrogen removal processes the CO composition in theC1-containing stream may be further enriched.

In various embodiments, the process may include multiple water gas shiftprocesses 520, 530 in combination with multiple hydrogen removalprocesses 540, 550, 560. As shown in FIG. 5, the C1-containing gaseoussubstrate may first be passed from a reforming process 510 to a watergas shift process 520 via piping means 511 to convert at least a portionof the CH₄ to a syngas stream comprising CO and H₂. This gas stream maythen be passed to one or more further water gas shift process 530 viapiping means 521 and be fed to the one or more hydrogen removal process540 via piping means 531 to separate at least a portion of the hydrogen541 from the gas stream. This stream may then be passed to one or morefurther hydrogen removal process 560 via piping means 542. The streamfrom the one or more further hydrogen removal process 560 may be sent tothe bioreactor 580 via piping means 561 for fermentation. At least aportion of the substrate not sent to the bioreactor may optionally besent to a subsequent hydrogen removal process 550 via piping means 562and optionally a further hydrogen removal process 590 via piping means551, which ultimately may be sent to the inoculation reactor 570, viapiping means 591, to produce an inoculum.

In various instances, the bioreactor 580 receives the gaseous substrateand produces one or more fermentation product 581. Optionally, the tailgas from both the inoculation reactor 570 and the bioreactor 580 can bepassed back to the reforming process 510 via separate piping means 572,582 and/or a blended stream 578.

In the various embodiments, the inoculation reactor 570 and thebioreactor 580 are configured in a step-wise manner, whereby theinoculation reactor 570 ferments a CO-rich C1-containing gaseoussubstrate to produce an inoculum, which is then fed to the bioreactor580 via piping means 571. By utilizing this inoculum in the bioreactor580, product selectivity and stability of the fermentation process isimproved.

It is to be understood, that whilst FIG. 3, FIG. 4 and FIG. 5 arerepresentations of an integration with a hydrogen production process thecurrent application is not to be limited to integration with a hydrogenproduction process.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed to limit its scope in any way.

Example 1

This example demonstrates the comparative performance of two reactorsprovided with a gaseous substrate comprising 68 vol. % H₂, 3.8 vol. %CO, 26 vol. % CO₂ and 1 vol. % N₂, an 18:1 molar ratio of H₂:CO. Theonly difference in the operating parameters of the two reactors was theconditions under which the inoculum for each reactor was produced. FIG.6a and FIG. 6b show metabolite and gas profiles in a first bioreactorthat received inoculum produced under CO-rich conditions. FIG. 7a andFIG. 7b show metabolite and gas profiles in a second bioreactor thatreceived inoculum produced under H₂ rich conditions. Both reactorsconsume H₂, CO, and CO₂ with similar efficiency, but the reactor thatreceived an inoculum from an H₂ rich inoculation reactor (FIG. 7a ) hasreduced selectivity to ethanol.

Example 2

This example demonstrates the comparative performance of reactorsprovided with inoculum from inoculation reactors operated underdiffering gas conditions. FIG. 8a and FIG. 8b show the metabolite andgas profiles of a fermentation inoculated with a culture received froman inoculation produced with the following gas composition: 48 vol. %H₂, 40 vol. % CO, 2 vol. % CO₂, and 10 vol. % N₂. FIG. 9a and FIG. 9billustrate the metabolite and gas profiles of a fermentation inoculatedwith a culture received from an inoculation produced under CO-richconditions. The ethanol selectivity demonstrated by the reactor fed byCO-rich gas inoculation reactor (FIG. 9a ) is much higher than that ofthe reactor that received an inoculum from an H₂ rich inoculationreactor (FIG. 8a ).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein. The reference to any prior art in this specification is not, andshould not be taken as, an acknowledgment that that prior art forms partof the common general knowledge in the field of endeavor in any country.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. The term “consistingessentially of” limits the scope of a composition, process, or method tothe specified materials or steps, or to those that do not materiallyaffect the basic and novel characteristics of the composition, process,or method. The use of the alternative (e.g., “or”) should be understoodto mean either one, both, or any combination thereof of thealternatives. As used herein, the term “about” means±20% of theindicated range, value, or structure, unless otherwise indicated.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For example, any concentration range,percentage range, ratio range, integer range, size range, or thicknessrange is to be understood to include the value of any integer within therecited range and, when appropriate, fractions thereof (such as onetenth and one hundredth of an integer), unless otherwise indicated.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein. Variationsof those preferred embodiments may become apparent to those of ordinaryskill in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A process comprising: a. producing a syngas comprising CO and H₂; b.passing the syngas to a hydrogen separation zone to produce a CO richgaseous substrate and an H₂ rich gaseous substrate; c. passing the COrich gaseous substrate to an inoculation reactor comprising a culture ofat least one C1-fixing microorganism in a liquid nutrient medium; d.fermenting the CO-rich gaseous substrate to produce an inoculum; e.passing at least a portion of the inoculum to at least one bioreactor,the bioreactor system comprising at least one bioreactor containing aculture of at least one C1-fixing microorganism in a liquid nutrientmedium; f. passing the H₂ rich gaseous substrate to at least onebioreactor; and g. fermenting the H₂ rich C1-containing gaseoussubstrate to produce at least one fermentation product.
 2. The processof claim 1 wherein the syngas is passed to a water gas shift reactor toconvert at least a portion of the CO and H₂ is converted to CO₂ andwater and then passing the gaseous substrate exiting the water gas shiftreactor to the hydrogen separation zone.
 3. The process of claim 2 wherethe gaseous substrate exiting the water gas shift reactor is passed to asecond water gas shift reactor and the exit stream from the second watershift reactor is passed to the hydrogen separation zone.
 4. The processof claim 1 where the hydrogen separation zone comprises a membraneseparation reactor.
 5. The process of claim 4 where at least a portionof the H₂ rich gaseous substrate from the membrane separation reactor ispassed to a pressure swing adsorption (PSA) reactor to produce ahydrogen stream and a CO and H₂ gaseous substrate which is passed to thebioreactor.
 6. The process of claim 1 where the hydrogen separation zoneis a PSA reactor.
 7. The process of claim 1, wherein the CO-rich gaseoussubstrate comprises H₂ at an H₂:CO molar ratio of less than 1:1.
 8. Theprocess of claim 1, wherein the CO-rich gaseous substrate comprises H₂at an H₂:CO molar ratio of less than 0.5:1.
 9. The process of claim 1,wherein the CO-rich gaseous substrate comprises H₂ at an H₂:CO molarratio between 0.02:1 to 1:1.
 10. The process of claim 1, wherein the H₂rich gaseous substrate comprises H₂ at an H₂:CO molar ratio of at least1.1:1.
 11. The process of claim 1, wherein the H₂ rich gaseous substratecomprises H₂ at an H₂:CO molar ratio between 1.1:1 to 6:1.
 12. Theprocess of claim 1, wherein the C1-fixing microorganism is acarboxydotrophic bacterium.
 13. The process of claim 12, wherein thecarboxydotrophic bacterium is selected from the group consisting ofMoorella, Clostridium, Ruminococcus, Acetobacterium, Eubacterium,Butyribacterium, Oxobacter, Methanosarcina, and Desulfotomaculum. 14.The process of claim 13, wherein the carboxydotrophic bacterium isClostridium autoethanogenum.
 15. The process of claim 18, wherein thesyngas is produced from steam methane reforming, steam naphta reforming,petroleum coke gasification, dry methane reforming and mixtures thereof.16. The process of claim 11, wherein the at least one fermentationproduct is selected from ethanol, acetate, butanol, butyrate,2,3-butanediol, 1,3-butanediol, lactate, butene, butadiene, methyl ethylketone, ethylene, acetone, isopropanol, lipids, 3-hydroxypropionate,isoprene, fatty acids, 2-butanol, 1,2-propanediol, 1-propanol,monoethylene glycol, isobutene, C6-C14 alcohols, and mixtures thereof.17. The process of claim 16, wherein the one or more fermentationproduct is further converted to at least one component of diesel fuel,jet fuel, gasoline, propylene, nylon 6-6, rubber, and/or resins.
 18. Theprocess of claim 16, wherein at least one fermentation product ismicrobial biomass.
 19. The process of claim 18, wherein the microbialbiomass is processed to produce at least one component of animal feed.